A flow cytometer is an instrument for measuring the fluorescence and light-scattering of biological cells and other microscopical particles. Carried by a laminar flow of water with a cross-section of the order of 100 .mu.m, the cells pass one-by-one through the focus of an intense source of excitation light. As a cell passes through this focus, it emits a short pulse of fluorescence as well as a pulse of scattered light. Thus, one can measure the cellular content of a constituent, such as DNA, which has been labelled with a fluorescent dye, as well as its size, which is determined from the light-scattering intensity. The excitation light source can be a continuous wave (cw) laser or some other high intensity light source having a constant intensity, such as a high pressure arc lamp containing either mercury or xenon. By means of dichroic mirrors, the fluorescence can be split into different spectral components which are measured by separate detectors. The scattered light may be measured by separate detectors at different scattering angles to provide information, not only on cell size, but also on structural features of the cells.
The fluorescence sensitivity, or fluorescence detection limit, of a flow cytometer is defined as the smallest amount of fluorescent material per cell that can be detected by the instrument. Likewise, the light-scattering sensitivity, or light-scattering detection limit, is defined as the smallest cell of a given composition that can be detected. The detection limit is determined by the size of the signal, as well as of the level of background, or noise, on which the signal is superimposed. This noise has two principally different sources: a) electrical noise from the electronics used to amplify and transmit the pulses from the light detectors, and b) optical noise which is due to the constant background of light caused by imperfect filters and fluorescence from lenses and other optical components. Whereas the electrical noise in flow cytometers can be reduced to an insignificant level, there is a principal limit to the optical noise. Thus, even if the power consumption of a light source is perfectly constant, the number of photons, n, emitted within a given period of time will fluctuate with a standard deviation, s, given by Equation 1: EQU s=n.sup.1/2 ( 1)
and the relative standard variation, cv, in the measurement of s is given by Equation 2: EQU s=n.sup.-1/2 ( 2)
This fluctuation of the light intensity, which we shall denote "optical noise", is a consequence of the stochastic nature of the process of light emission and is true for all light sources, including the fluorescent cell and the various sources of optical background in the flow cytometer. The optical noise thus puts a principal limit to any measurement of light intensity. Only by increasing n, that is either by increasing the light intensity or by increasing the observation period, can this limit be reduced. In the flow cytometer the observation period is limited by the passage time of the cell through the excitation focus.
Since the signal increases in proportion to the excitation intensity, while the optical noise, according to Equation 1, increases in proportion to the square root of the intensity, the signal-to-noise ratio will increase in proportion to the square root of the excitation intensity, and the detection limit will decrease in the same proportion.